Sensor platform, apparatus incorporating the platform and process using the platform

ABSTRACT

A sensor platform for use in sample analysis comprises a substrate ( 30 ) of refractive index (n 1 ) and a thin, optically transparent layer ( 32 ) of refractive index (n 2 ) on the substrate, (n 2 ) is greater than (n 1 ). The platform incorporates one or multiple corrugated structures in the form of periodic grooves ( 31 ), ( 33 ), which defines one or more sensing areas each for one or more capture elements. The grooves are so profiled, dimensioned and oriented that when coherent light is incident on the platform it is diffracted into individual beams or diffraction order resulting in reduction of the transmitted beam and an abnormal high reflection of the incident light thereby creates an enhanced evanescent field at the surface of the or each sensing area. The amplitude of this field at the resonant condition is greater by an order of approximately 100 than the field of prior art platforms so that the luminescence intensity created from samples on the platform is also increased by a factor of 100. Also disclosed are an apparatus incorporating the platform and a method of using the platform. Further increases of amplitude have been detected by using light having a linear component which gives rise to TM excitation and/or irradiating the platform from the substrate side.

This application is a continuation-in-part of pending U.S. patentapplication Ser. No. 09/609,846, filed Jul. 5, 2000.

This invention relates generally to the field of analysing samples andhas particular, but not exclusive, application in the field of affinitysensing for example that known generally as DNA, protein, peptide andantibody chip technology. One aspect of the invention is concerned witha sensor platform which can be used to analyse samples. Another aspectof the invention is concerned with an apparatus which makes use of thesensor platform. A further aspect of the invention is concerned with theprocess for analysing samples which makes use of the platform.

Techniques for analysing two-dimensional arrays of samples are known.One such technique is known as an ELISA assay and is based upon theintense biochemical reaction between antibodies and antigens. Specialmono or polyclonal antibodies are immobilised on substrates and reactwith complimentary species. Fluorophore labelled markers are added,activated via enzyme-linked antibodies, and the samples are irradiatedwith light in order to induce fluorescence. The fluorescence is detectedand the intensity of the fluorescence is indicative of the affinityreaction.

Another known technique is that described in WO98/27430. In this a largenumber of different species are immobilised in an array on a substrate.The species are immobilised on the substrate by photolithographicalmeans. Fluorophore labelled markers are added to the species. A sampleis prepared and reacted with the immobilised species and the whole chipis scanned with a focused laser bean. Alternatively a sample is preparedand modified with fluorophore labelled markers and ated with theimmobilised species and the whole chip is scanned with a focussed laserbeam. The fluorescent signals are detected by photodetectors and a 2Dpattern is produced. Changes in this pattern between individual samplesprovide an indication of differences in gene expression and thereforeprovides information about pharmacology and toxicology.

Another known technique is that based on evanescent wave sensors. Thesemake use of coherent laser light which is trapped in a very thin layerand creates so-called evanescent electromagnetic fields which extendsfor a small distance outside the actual physical sensor. This field caninteract with molecules attached to the surface of the sensor. Thisevanescent excitation or interaction is limited to a region very closeto the vicinity of the waveguide, typically 0.5 microns for visiblelight from the surface. The evanescent fields remain localised spatiallyand do not transfer their stored energy to other regions. Theinteraction of the laser light with the molecules can be used in anumber of different ways. These include:

-   1 Detection of luminescence induced by the evanescent field.-   2 Detection of changes in refractive index which occur when    molecules of a sample bind to capture molecules.-   3 Detection of surface plasmon resonance.

One particular sensor which uses an evanescent field is known as aplanar waveguide sensor. The planar waveguide sensor comprises a planarsubstrate having formed thereon a thin wave guiding layer. Part of thewave guiding layer incorporates a grating onto which laser light isincident and from which the laser light is launched so that itpropagates through the waveguide layer to a sensing region remote fromthe grating. The waveguide sensor can be either used in a mass sensitivemode (cf. 2, 3 above), or with superior sensitivity in combination withluminescence excitation and detection (cf. 1 above). Capture moleculesare immobilised on the sensing area and the analyte (sample) is thenbrought into contact with the sensing area/capture molecules in thepresence of added labelled molecules with similar affinity(competition). Alternatively, analyte molecules may bind to immobilisedcapture molecules and fluorescence labels are introduced by reaction ofa further labelled species with the captured analyte molecules. Laserlight launched into the waveguiding layer leads to evanescent excitationof the fluorophores which then allows the quantification of the analyte.The emitted fluorescence is detected and the intensity of thefluorescence provides an indication of the interaction that has occurredbetween affinity partners present in the analyte and the immobilisedcapture molecules. It should be noted that in this type of arrangementthe laser radiation propagates inside the waveguide over relatively longdistances and the coupling grating and the sensing areas aregeometrically separated. (See WO 95/33197 and WO 95/33198).

EP-0 455 067 A2 describes a planar waveguide sensor exploiting thedetecting principle of refractive index changes. The platform shallowgrooves formed over the entire platform couple polarised, coherent lightinto the transparent waveguiding layer where it is coupled out aftersome distance. The angle of the outcoupled beam changes when analytemolecules bind to capture molecules.

Another example of the refractive index type is given in U.S. Pat. No.5,738,825. The platform contains individual gratings being in contactwith the wells of a microtiter plate.

EP 178 083 discloses Surface Plasmon Resonance (SPR) in which the energyof incoming photons is converted to electrical energy as a surfaceplasmon wave. The sensor architecture requires a metal layer in contrastto the platform of the present invention, and the amount of reflectedlight at the critical angle is, or approximates to, zero in contrastwith the present invention in which the reflected intensity reachesalmost 100%.

All the above techniques suffer from various disadvantages. Some arevery slow because each sample has to be excited individually. Otherssuch as the planar waveguide allow excitation of more than one sample ata time, but do not provide entirely reliable results because offluorescence crosstalk between different capture elements and locallyvarying excitation light intensities due to losses of the waveguides andlocal variations of coupled power due to variations of grating couplingefficiencies.

The present invention is concerned with a technique which allowsmultiple samples to be analysed simultaneously in an extremelysensitive, reliable, and quantitative-manner.

In contrast to planar waveguide sensors, the present invention shows noluminescence crosstalk and local light intensities are well defined. Thepresent invention allows true multiplexing, i.e. the transducer requiresno stacked substructure (as is the case for planar waveguides) and canbe seen as a universal platform, where, depending on the requirements,size and number of recognition elements can be varied within thetechnical feasible limitations, without requiring changes in the chipstructure (corrugated areas and sensing areas are not separated as isthe case for planar waveguides). In addition, the invention deliversabout 100 fold stronger luminescence intensities compared to priorepifluorescence techniques. The experimental set-up is very simple andrequires solely a simple adjustment of the angle of the incident lightbeam. The transducers described in the present invention, can be easilyadapted to conventional fluorescence microscopes, confocal microscopes,and laser scanners. Furthermore, for transducers with a broad resonancewidth (defined as Full Width at Half Maximum, FWHM) and a resonanceposition at or close to normal incidence, angle adjustments areobsolete.

The production process of the platform is relatively simple (cheap) andthe performance of existing systems (i.e. fluorescence scanners,microscopes, fluorescence microtiter plate readers, . . . ) can beeasily increased by modest modifications of the respective set-ups.

According to a first aspect of the present invention there is provided aplatform for use in sample analysis comprising an optically transparentsubstrate having a refractive index (n₁), a thin, optically transparentlayer, formed on one surface of the substrate, said layer having arefractive index (n₂) which is greater than (n₁), said platformincorporating therein one or multiple corrugated structures comprisingperiodic grooves which define one or multiple sensing areas or regions,each for one or multiple capture elements, said grooves being soprofiled, dimensioned and oriented that either

-   a) coherent light incident on said platform is diffracted into    individual beams or diffraction orders which interfere resulting in    reduction of the transmitted beam and an abnormal high reflection of    the incident light thereby generating an enhanced evanescent field    at the surface of the one or multiple sensing areas; or-   b) coherent and linearly polarised light incident on said platform    is diffracted into individual beams or diffraction orders which    interfere resulting in almost total extinction of the transmitted    beam and an abnormal high reflection of the incident light thereby    generating an enhanced evanescent field at the surface of the one or    multiple sensing areas.

According to a second aspect of the present invention there is provideda platform comprising an optically transparent substrate having arefractive index (n1), a thin, optically transparent layer, formed onone surface of the substrate, said layer having a refractive index (n2)which is greater than (n1), said platform incorporating in thetransparent layer a corrugated structure substantially over the entireplatform, or multiple separate corrugated structures arranged on theplatform, said structures comprising substantially parallel periodicgrooves which are mono- or multi-diffractional which grooves representone or multiple sensing areas or regions, wherein

-   (a) the depth of the grooves is in the range of 3 nm to the    thickness of the optically transparent layer,-   (b) the thickness of the optically transparent layer is in the range    of 30 to 1000 nm,-   (c) the period of the corrugated structure is in the range of 200 to    1000 nm,-   (d) the ratio of groove depth to the thickness of the optically    transparent layer is in the range of 0.02 to 1, and-   (e) the ratio of groove width to the period of the grooves is in the    range of 0.2 to 0.8. The arrangement may be such that, in use, the    grooves are so profiled, dimensioned and oriented that either-   a) coherent light incident on the platform is diffracted into    individual beams or diffraction orders which interfere resulting in    reduction of the transmitted beam and an abnormal high reflection of    the incident light thereby generating an enhanced evanescent field    at the surface of the one or multiple sensing areas; or-   b) coherent and linearly polarised light incident on said platform    is diffracted into individual beams or diffraction orders which    interfere resulting in almost total extinction of the transmitted    beam and an abnormal high reflection of the incident light thereby    generating an enhanced evanescent field at the surface of the one or    multiple sensing areas.

As used herein, orientation is understood to mean that the electricfield vector of the linearly polarised light is parallel orperpendicular to the grooves. As used herein, coherent light isunderstood to mean that the coherence length of the radiation, i.e. thespatial extent to which the incident beam has a defined phase relation,is large compared to the thickness of the platform.

The evanescent field decays exponentially within wavelength dimensionsof the incident beam (less than 1 μm).

An important aspect of the present invention is the use of a platform inwhich so-called evanescent resonance can be created. Abnormal reflectionis a phenomenon which has been described theoretically in the prior artfor example in a paper entitled “Theory and applications of guided moderesonance filters” by S S Wang and R Magnusson in Applied Optics, Vol.32, No 14, 10 May 1993, pages 2606 to 2613 and in a paper entitled“Coupling gratings as waveguide functional elements” by O. Parriaux etal, Pure & Applied Optics 5, (1996) pages 453-469. As explained in thesepapers resonance phenomena can occur in planar dielectric layerdiffraction gratings where almost 100% switching of optical energybetween reflected and transmitted waves occurs when the grooves of thediffraction grating have sufficient depth and the radiation incident onthe corrugated structure is at a particular angle. In the presentinvention this phenomenon is exploited in the sensing area of theplatform where that sensing area includes diffraction grooves ofsufficient depth and light is caused to be incident on the sensing areaof the platform at an angle such that evanescent resonance occurs in titsensing region. This creates in the sensing region an enhancedevanescent field which is used to excite samples under investigation. Itshould be noted that the 100% switching referred to above occurs withparallel beam and linearly polarised coherent light and the effect of anenhanced evanescent field can also be achieved with non-polarised lightof a non-parallel focussed laser beam.

At resonance conditions the individual beams interfere in such a waythat the transmitted beam is cancelled out (destructive interference)and the reflected beam interferes constructively giving rise to abnormalhigh reflection.

By choosing appropriate parameters for the above mentioned corrugatedlayer structure the excitation energy remains highly localized. Suchstructures are described in the literature as photonic band gapstructures, materials with periodic spatial variations of theirrefractive index such that electromagnetic radiation cannot propagate inany direction. Photonic bandgap structures allow highly localized modesto appear, see e.g. the paper entitled “Localisation of One PhotonStates” by C. Adlard, E. R. Pike and S. Sarkar in Physical ReviewLetters, Vol. 79, No 9, pages 1585-87 (1997). Such structures exhibitextremely large propagation losses corresponding to a mode localisationin the 1 μm regime.

The platform of the present invention can be considered as opticallyactive in contrast to optically passive platforms constructed from e.g.a glass or polymer. Here, optically active means increasing theelectromagnetic field of the excitation beam by energy confinement.

The substrate of the platform may be formed from inorganic materialssuch as glass, SiO₂, quartz, Si. Alternatively the substrate can beformed from organic materials such as polymers preferably polycarbonate(PC), poly(methyl methacrylate) (PMMA), polyimide (PI), polystyrene(PS), polyethylene (PE), polyethylene terephthalate (PM) or polyurethane(PU). These organic materials are especially preferred for point-of-care(POC) and personalized medical applications since glass is not acceptedin such an environment Plastics substrates can be structured (embossed)much more easily than glass. In one example the substrate is formed fromglass.

The optically transparent layer may be formed from inorganic material.Alternatively it can be formed from organic material. In one example theoptically apart layer is a metal oxide such as Ta₂O₅, TiO₂, Nb₂O₅, ZrO₂,ZnO or HfO₂. The optically transparent layer is non-metallic.

Alternatively the optically transparent layer can be made of organicmaterial such as polyamide, polyimide, polypropylene (PP), PS, PMMA,polyacryl acids, polyacryl ethers, polythioether,poly(phenylenesulfide), and derivatives thereof (see for example S S.Hardecker et al., J. of Polymer Science B: Polymer Physics, Vol. 31,1951-63, 1993).

The depth of the diffraction grooves may be in the range 3 nm to thethickness of the optically transparent layer and preferably 10 nm to thethickness of the optically transparent layer e.g. 30 nm to the thicknessof the optically transparent layer. The thickness of the opticallytransparent layer be in the range 30 to 1000 nm, e.g. 50 to 300 nm,preferably 50-200 nm, the period of the corrugated structure may be inthe range 200 to 1000 nm, e.g. 200 to 500 nm, preferably 250-500 nm, theratio of the groove depth to the thickness of the optically transparentlayer may lie in the range 0.02 to 1 e.g. 0.25 to 1, preferably 0.3 to0.7, and the ratio of the grooves width to the period of the grooves(“duty-cycle”) may lie in the range 0.2 to 0.8, e.g. 0.4 to 0.6.

The grooves may be generally rectangular in cross-section.Alternatively, the grooves may be sinusoidal or of saw toothcross-section. The surface structure may be generally symmetrical.Preferred geometries include rectangular, sinusoidal, and trapezoidalcross-sections. Alternatively, the grooves may be of saw toothcross-section (blazed grating) or of other asymmetrical geometry. Inanother aspect the groove depth may vary, e.g. in periodic modulations.

The platform may be square or rectangular and the grooves may extendlinearly along the platform so as to cover the surface. Alternativelythe platform may be disc shaped and the grooves may be circular orlinear.

The grooves may be formed on a surface of the substrate. Alternativelythe grooves may be formed on a surface of the optically transparentlayer. As a further alternative, grooves may be formed both on thesurface of the substrate which is the interface and on the surface ofthe optically transparent layer.

The corrugated surface of a single sensing area may be optimized for oneparticular excitation wavelength and for one particular type ofpolarisation. By appropriate means, e.g. superposition of severalperiodic structures which are parallel or perpendicular one withanother, periodic surface reliefs can be obtained that are suitable formultiple wavelength use of the platform “multicolour” applications).Alternatively, individual sensing areas on one platform may be optimizedfor different wavelengths and/or polarization orientations.

The surface of the optically transparent layer may include one or aplurality of corrugated sensing areas which each may carry one or aplurality of capture elements.

Each capture element may contain individual and/or mixtures of capturemolecules which are capable of affinity reactions. The shape of anindividual capture element may be rectangular, circular, ellipsoidal, orany other shape. The area of an individual capture element is between 1μm² and 10 mm², e.g. between 20 μm² and 1 mm² and preferably between 100μm² and 1 mm². The capture elements may be arranged in a regular twodimensional array. The center-to-enter (ctc) distance of the captureelements may be between 1 μm and 1 mm, e.g. 5 μm to 1 mm, preferably 10μm to 1 mm.

The number of capture elements per sensing area is between 1 and1,000,000, preferably 1 and 100,000. In another aspect, the number ofcapture elements to be immobilized on the platform may not be limitedand may correspond to e.g. the number of genes, DNA sequences, DNAmotifs, DNA micro satelites, single nucleotide polymorphisms (SNPs),proteins or cell fragments constituting a genome of a species ororganism of interest, or a selection or combination thereof. In afurther aspect, the platform of this invention may contain the genomesof two or more species, e.g. mouse and rat.

The platform may include an adhesion promoting layer disposed at thesurface of the optically transparent layer in order to enableimmobilisation of capture molecules. The adhesion promoting layer mayalso comprise a microporous layer (ceramics, glass, Si) for furtherincreasing assay and detection efficacy or of gel layers which eithercan be used as medium for carrying out the capture elementimmobilisation and sample analysis, thereby further increasing the assayand detection efficacy, or which allow separation of analyte mixtures inthe sense of gel electrophoresis. The platform may be formed with aplurality of sensing areas or regions, each having its own diffractivegrooves.

A feature of the platform of this invention is that light energyentering the optically transparent layer is diffracted out of the layerimmediately due to the nature of the corrugated platform. Therefore noor negligible waveguiding occurs. Typically the propagation distance is100 μm or less, preferably 10 μm or less. This is a very surprisinglyshort distance. The propagation distance is the distance over which theenergy of the radiation is reduced to 1/e.

A third aspect of the invention provides apparatus for analysing samplescomprising a platform according to said first or second aspect, meansfor generating a light beam and for directing the beam so that it isincident upon the platform at an angle which causes evanescent resonanceto occur in the platform to thereby create an enhanced evanescent fieldin the sensing area of the platform, and means for detecting acharacteristic of a material disposed on the sensing area of theplatform. The range of angles suitable for crating a resonance conditionis limited by the angle of total reflection for incident light on theplatform. Preferred angles are less than 45°, e.g. 30° or less, e.g. 20°to 10° or below, e.g. 0.1° to 9.9°. The may equal or approximate normalincidence. The light generating means may comprise a laser for emittinga coherent laser beam. Other suitable light sources include dischargelamps or low pressure lamps, e.g. Hg or Xe, where the emitted spectrallines have sufficient coherence length, and light-emitting diodes (LED).The apparatus may also include optical elements for directing the laserbeam so that it is incident on the platform at an angle θ, and elementsfor shaping the plane of polarisation of the coherent beam, e.g. adaptedto transmit linearly-polarised light. The angle θ may be defined by theexpression sin θ=n−λ/Λ where Λ is a period of the diffractive grooves, λis the wavelength of the incident light and n is the effectiverefractive index of the optically transparent layer.

Examples of lasers that may be used are gas lasers, solid state lasers,dye lasers, semiconductor lasers. If necessary, the emission wavelengthcan be doubled by means of non-linear optical elements. Especiallysuitable lasers are argon ion lasers, krypton ion lasers, argon/kryptonion lasers, and helium/neon lasers which emit at wavelengths between 275and 753 nm. Very suitable are diode lasers or frequency doubled diodelasers of semiconductor material which have small dimensions and lowpower consumption.

Another appropriate type of excitation makes use of VCSEL's (verticalcavity surface-emitting lasers) which may individually excite therecognition elements on the platform.

The detecting means may be arranged to detect luminescence such asfluorescence. Affinity partners can be labelled in such a way thatFörster fluorescence energy transfer (FRET) can occur upon binding ofanalyte molecules to capture molecules. The maximum of the luminescenceintensity might be slightly shifted relative to the position of highestabnormal reflection depending on the refractive index values of thelayer system and the corresponding Fresnel Coefficients.

The samples may be used either undiluted or with added solvents.Suitable solvents include water, aqueous buffer solutions, proteinsolutions, natural or artificial oligomer or polymer solutions, andorganic solvents. Suitable organic solvents include alcohols, ketones,esters, aliphatic hydrocarbons, aldehydes, acetonitrile or nitriles.

Solubilisers or additives may be included, and may be organic orinorganic compounds or biochemical reagents such asdiethylpyrocarbonate, phenol, formamide, SSC (sodium citrate/sodiumchloride), SDS (Sodiumdodecylsulfate), buffer reagents, enzymes, reversetranscriptase, RNAase, organic or inorganic polymers.

The sample may also comprise constituents that are not soluble in thesolvents used, such as pigment particles, dispersants and natural andsynthetic oligomers or polymers.

The luminescence dyes used as markers may be chemically or physically,for instance electrostatically, bonded to one or multiple affinitybinding partners (or derivatives thereof) present in the analytesolution and/or attached to the platform. In case of naturally-occurringoligomers or polymers such as DNA, RNA, saccharides, proteins, orpeptides, as well as synthetic oligomers or polymers, involved in theaffinity reaction, intercalating dyes are also suitable. Luminophoresmay be attached to affinity partners present in the analyte solution viabiological interaction such as biotin/avidin binding or metal complexformation such as HIS-tag coupling.

One or multiple luminescence markers may be attached to affinitypartners present in the analyte solution, to capture elementsimmobilized on the platform, or both to affinity partners present inanalyte solution and capture elements immobilized at the platform, inorder to quantitatively determine the presence of one or multipleaffinity binding partners. The spectroscopic properties of theluminescence markers may be chosen to match the conditions for FörsterEnergy Transfer or Photoinduced Electron Transfer. Distance andconcentration dependent luminescence of acceptors and donors may then beused for the quantification of analyte molecules.

Quantification of affinity binding partners may be based onintermolecular and/or intramolecular interaction between such donors andacceptors bound to molecules involved in affinity reactions.Intramolecular assemblies of luminescence donors and acceptorscovalently linked to affinity binding partners, Molecular Beacons (S.Tyagi et al., Nature Biotechnology 1996, 14, 303-308) which change thedistance between donor and acceptor upon affinity reaction, may also beused as capture molecules or additives for the analyte solution. Inaddition, pH and potentially sensitive luminophores or luminophoressensitive to enzyme activity may be used, such as enzyme mediatedformation of fluorescing derivatives.

Transfluorospheres or derivatives thereof may be used for fluorescencelabelling, and chemi-luminescent or electroluminescent molecules may beused as markers.

Luminescent compounds having luminescence in the range of from 400 nm to1200 nm which are functionalised or modified in order to be attached toone or more of the affinity partners, such as derivatives of

-   -   polyphenyl and heteroaromatic compounds    -   stilbenes,    -   coumarines,    -   xanthene dyes,    -   methine dyes,    -   oxazine dyes,    -   rhodamines,    -   fluoresceines,    -   coumarines, stilbenes,    -   pyrenes, perylenes,    -   cyanines, oxacyanines, phthalocyanines, porphyrines,        naphthalopcyanines, azobenzene derivatives, distyryl biphenyls,    -   transition metal complexes e.g. polypyridyl/ruthenium complexes,        tris(2,2′-bipyridyl)ruhenium chloride,        tris(1,10-phenanthroline)ruthenium chloride,        tris(4,7-diphenyl-1,10-phenanthroline) ruthenium chloride and        polypyridyl/phenazine(ruthenium complexes, such as        octaethyl-platinum-porphyrin, Europium and Terbium complexes may        be used as luminescence markers.

Suitable for analysis of blood or serum are dyes having absorption andemission wavelength in the range from 400 nm to 1000 nm. Furthermoreluminophores suitable for two and three photon excitation can be used.

Dyes which are suitable in this invention may contain functional groupsfor covalent bonding, e.g. fluorescein derivatives such as fluoresceinisothiocyanate.

Also suitable are the functional fluorescent dyes commercially availablefrom Amersham Life Science, Inc. Texas. and Molecular Probes Inc.

Other suitable dyes include dyes modified with deoxynucleotidetriphosphate (dNTP) which can be enzymatically incorporated into RNA orDNA strands.

Further suitable dyes include Quantum Dot Particles or Beads (QuantumDot Cooperation, Palo Alto, Calif.) or derivatives thereof orderivatives of transition metal complexes which may be excited at oneand the same defined wavelength, and derivatives show luminescenceemission at distinguishable wavelengths.

Analytes may be detected either via directly bonded luminescencemarkers, or indirectly by competition with added luminescence markedspecies, or by concentration-, distance-, pH-, potential- or redoxpotential-dependent interaction of luminescence donors andluminescence/electron acceptors used as markers bonded to one and/ormultiple analyte species and/or capture elements. The luminescence ofthe donor and/or the luminescence of the quencher can be measured forthe quantification of the analytes.

In the same manner affinity partners can be labelled in such a way thatelectron transfer or photoinduced electron transfer leads to quenchingof fluorescence upon binding of analyte molecules to capture molecules.

Appropriate detectors for luminescence include CCD-cameras,photomultiplier tubes, avalache photodiodes, photodiodes, hybridphotomultiplier tubes.

The detection means can be arranged to detect in addition changes inrefractive index.

The incident beam may be arranged to illuminate the sensing area or allsensing areas on one common platform. Alternatively the beam can bearranged to illuminate only a small sub-area of the sensing area to beanalysed and the beam and/or the platform may be arranged so that theycan undergo relative movement in order to scan the sensing area of theplatform.

Accordingly the detecting means may be arranged in an appropriate way toacquire the luminescence signal intensities of the entire sensing areain a single exposure step. Alternatively the detection and/or excitationmeans, may be arranged in order to scan the sensing areas stepwise.

The apparatus may include a cartridge for location against the sensingarea of the platform to bring a sample into contact with the sensingarea. The cartridge may contain further means in order to carry outsample preparation, diluting, concentrating, mixing, bio/chemicalreactions, separations, in a miniaturised format (see WO 97/02357). Theapparatus may include a microtiter type device for containing aplurality of samples to be investigated.

A fourth aspect of the present invention provides a process foranalysing a sample or samples which comprises bringing the sample intocontact with the sensing area of a platform according to said first orsecond aspect, irradiating the platform with a light beam such thatevanescent resonance is caused to occur within the sensing area of theplatform and detecting radiation emanating from the sensing area. Themethod may comprise adding fluorescent inducing material to the samplesunder investigation and sensing fluorescence induced in said samples byexcitation of the samples by the enhanced evanescent field.Alternatively the method may comprise adding fluorescence inducing orquenching material to the samples under investigation and/or transfer ofthe samples under investigation into fluorescing or quenchingderivatives and sensing fluorescence induced by said samples bound atthe sensing platform by excitation with the enhanced evanescent field.

It is believed to be a novel and inventive concept to provide a sensorplatform in which each sensing area or region has attached thereto morethan one type of capture element or molecule. This concept applieswhether the platform is designed for evanescent resonant ode or a moreconventional mode such as waveguiding. Thus, according to another aspectof the present invention there is provided a platform for use in asample analysis, said platform having one or more sensing areas orregions, each for receiving a capture element or elements which when theplatform is irradiated with coherent light can interact to provide anindication of an affinity reaction, wherein each capture elementincludes two or more types of capture molecule.

The above-described embodiments of this invention contemplate lightpolarised parallel with the longitudinal axis of the grooves of theplatform, giving rise to “TE” excitation, or light polarisedperpendicular to the longitudinal axis of the grooves of the platform,giving rise to “TM” excitation. Further, light may be incident eitheronto the corrugated, optically transparent, high refractive index layerside of the platform (“chip”) or onto the other side of the platform,i.e. onto the optically transparent substrate side.

The nature of polarisation and aspects of excitation of TE and TM modesare discussed in Guided Wave Optoelectronics, Ed. T. Tamir, 1988,Springer Verlag, specifically the Chapter entitled Theory of OpticalWaveguides, Author: H. Kogelnik, the contents of which are incorporatedherein by reference.

The present applicants have found that even greater amplification can beobtained by exploiting the abnormal reflection geometry provided by TMexcitation compared to TE excitation.

A further increase in sensitivity may be obtained when the lightincident on the platform is directed onto the optically transparentsubstrate. This may be attractive for e.g. fluorescence laser scanners.

In a further aspect, therefore, this invention provides a platform,apparatus or detection method as described herein adapted to TMexcitation.

It will be appreciated that varying degrees of polarisation of theincident light beam may be employed, depending e.g. on field ofapplication and resources available or required. The abnormal reflectionand/or fluorescence enhancement described herein is observed preferablyfrom the linearly polarised component of the incident light. Thus thelight incident on the platform may be e.g. substantially linearlypolarised or circularly or elliptically polarised.

Increases of signal intensity amplification have been detected by afactor of up to around 5 to 10 using TM excitation over TE excitation.

In a further aspect, this invention provides a platform, apparatus ordetection method as described herein wherein the light bean isirradiated onto the transparent substrate side of the platform. Theapplicants have found that several times greater signal intensity, e.g.a factor of 5 to 7, may be obtained when irradiating the platform ontothe substrate side instead of onto the corrugated, opticallytransparent, high refractive index layer side of the platform.

An increase in signal intensity amplification by a factor of around 50may be obtained by employing TM excitation with the light beam incidentonto the substrate side compared with TE excitation and the light beamincident onto the corrugated, optically transparent, high refractiveindex layer side of the platform.

The invention will be described now by way of example only withparticular reference to the accompanying drawings. In the drawings:

FIG. 1 is a schematic illustration of a quality control apparatus foranalysing the optical parameters and the evanescent resonance conditionof a platform in accordance with the present invention;

FIG. 2 is a schematic illustration of a sensor platform in accordancewith the present invention;

FIG. 3 is a schematic view showing the evanescent field profile inrelation to the platform;

FIGS. 4 a and 4 b are schematic views showing a chip cartridge;

FIG. 5 shows an array layout in one example of the present invention;

FIG. 6 shows schematically the layout used to measure fluorescencesaccording to one example of the invention;

FIG. 7 shows a comparison of results obtained by a prior art techniqueand the present invention;

FIG. 8 illustrates alternative forms of platform;

FIGS. 9 a to 9 c show fluorescence images obtained after incubation of30 pm PM analyte, regeneration, and 30 pM MM analyte using the presentplatform under resonance conditions as described in Example 5;

FIG. 10 shows fluorescence images and data obtained using the presentplatform under epifluorescence and resonance conditions as described inExample 6;

FIG. 11 shows angular distribution of fluorescence intensity with TMexcitation; and

FIG. 12 shows angular distribution of fluorescence intensity with TEexcitation.

The invention will be described in terms of the determination ofluminescence excited in samples. This determination involves the use ofa sensor platform which constitutes one aspect of the present invention,but it will be appreciated that the use of such a platform is notnecessarily restricted to the particular application to be described.Before describing the platform in detail a description will be given ingeneral terms of the way in which the platform can be used to determineluminescence of samples.

The following are definitions of terms which will be used in thedescription:

-   Platform: a whole transducer/chip containing one or a plurality of    sensing areas-   Sensing area: a whole corrugated area capable of creating an    evanescent field by a resonance effect and containing one or a    plurality of capture elements-   Capture element: an individual sensing spot containing one or a    variety of species of capture molecules-   Capture molecule: an individual molecule capable of an affinity    reaction-   Counts: signal intensity measured over a predetermined time    interval. This time interval is one second unless otherwise stated.    In the following examples all temperatures are in degrees Centigrade    and are uncorrected.

Referring to FIG. 1 a platform in accordance with an aspect of thepresent invention is shown at (10) and can receive coherent light from alaser (11), the laser light having been expanded by a set of lenses (12,14) which produce an expanded and parallel beam (16), and polarised by apolariser (18). As will be explained in more detail later, the platform(10) has a sensing area to which are attached capture molecules. Thewavelength of the light will typically be in the range UV to NIR range,preferably between 350 nm to 1000 nm.

The apparatus also includes a detector (20) which can detect lighttransmitted through the platform (10), a CCD camera (21) to detect thereflected light and a data processing unit (22).

In use of the apparatus a highly parallel, expanded, coherent, linearlypolarised, laser beam (16) is caused to be incident on the sensing areaof the platform (10) and light transmitted through the platform issensed by detector (20) and the reflected light is recorded by the CCDcamera (21). The diameter of the expanded excitation beam exceeds thesize of the platform (10). The angle of incidence of the beam on theplatform is adjusted by rotation of the platform until the detector (20)detects effectively no light being transmitted through the platform.This indicates the existence of a resonance position at which evanescentresonance is occurring in the sensing area of the platform. Under thiscondition, the reflected light intensity recorded by the camera (21)reaches a maximum and the data from the camera is acquired by the dataprocessing unit (22) for processing

Turning now to FIG. 2 of the drawings, an embodiment of the platform(10) comprises a glass substrate (30) into the top surface of which hasbeen etched a plurality of grooves (31). A layer of opticallytransparent metal oxide (32) is deposited on the upper surface of thesubstrate (30) and that layer (32) also has formed therein grooves (33).The substrate (30) can for example be formed from glass such as glassAF45 produced by Schott and typically has a thickness of 0.5 mm-1.0 mm.It will be appreciated that other organic or inorganic materials can beused for the substrate provided that it is optically transparent.

The optically transparent layer is a dielectric transparent metal oxidefilm such as Ta₂O₅ with a high refractive index of approximately 2.2 ata wavelength of 633 nm, i.e. significantly higher than the refractiveindex of the substrate. The thickness of this layer will typically be inthe range 50 to 200 nm or greater e.g. 50 to 300 nm. The corrugatedstructures (31) and (33) have a period in the range of 200-1000 nm, e.g.200 to 500 nm, typically 250-500 nm. The depth of the corrugatedstructures/diffraction grooves may be in the range 3 nm to the thicknessof the optically transparent layer, preferably 10 nm to the thickness ofthe optically transparent layer. The metal oxide can be any of a numberof examples such as Ta₂O₅, TiO₂, Nb₂O₅, ZrO₂, ZnO, or HfO₂.

In a platform such as that shown in FIG. 2, when a parallel beam ofpolarised laser light is incident thereon at a particular angle ofincidence, an effect known as abnormal reflection occurs within thelayer (32). When this effect occurs substantially no light istransmitted through the platform (10) and effectively all the light isreflected within the layer (32) so that the coherent laser light isconfined to the very thin layer (32) of metal oxide. The resulting highlaser field leaks partially out of the layer (32) creating an evanescentfield which evanescently excites fluorescent material which is on thesurface or in the close vicinity of the layer (32). It should be notedthat this resonance condition can be achieved only when diffractivegrooves (31, 33) having a particular depth or greater are employed andit should also be noted that the radiation losses of such a corrugatedstructure are very high so that effectively no waveguiding of anyelectromagnetic radiation in the preferred wavelength range occurswithin the layer of metal oxide (32). It is preferred that the depth ofthe grooves be at least 10 nm but the evanescent resonance starts tobuild up with shallower grooves. However, provided that the sale to beinvestigated is in the vicinity of the layer (32) at which the resonanceis created the enhanced evanescent field can be used to exciteluminescence such as fluorescence in the sample.

An important feature of the present platform is that the amplitude ofthis evanescent field at resonance position is significantly greaterthan that of the prior art arrangements (epifluorescence correspondingto off-resonance condition) by an order of approximately 100.

This means that the intensity of luminescence, e.g. fluorescence, whichcan be created from samples is also increased by a factor of 100. Thefunction of the platform can be viewed in terms of the diffractivestructure acting as a volume grating which diffracts light and that thediffractive beams interfere creating a resonance condition where thelight reflected from the first interface and light reflected from thetop interface that is the upper surface of the layer (32) interfereconstructively giving rise to reflection maxima. Under resonanceconditions, the laser energy is substantially confined to the thicknessof the thin layer (32) thereby increasing the electrical field strength.For a given laser wavelength and period of the corrugated structure, theresonance is angle-dependent. The angle dependent resonance typicallyhas a width at half maximum height (FWHM) of >0.1° preferably 0.5° orgreater e.g. 1.0° or greater. This resonance width is dependent upon thedepth of the grooves, the duty cycle and geometry of the corrugatedstructure. Compared to coupling behaviour of a waveguide grating, theFWHM of the resonance described is greater by many orders of magnitude.

It will be appreciated that the diffraction grooves (31, 33) can beformed on the platform by appropriate conventional techniques. One wayof achieving this is to etch the grooves by a photographical technique.In this, a photoresist composition is deposited on the surface of thesubstrate to a depth of approximately 1 μm, a periodic structurecorresponding to the groove formation is then written into the resisteither by two beam interferometry/holography or by use of a phase maskand then the resist is etched with a reactive ion etching techniqueusing argon gas and finally the residual photoresist material isstripped from the surface. This technique can be used for forming bothgrooves (31) and grooves (33). Other ways of incorporating thecorrugated structures include embossing, electron beam writing, laserablation, LIGA process.

In order to prepare a platform of the type described with reference toFIG. 2 so that it can be used in a measurement such as that illustratedin FIG. 6, a number of procedures should be followed.

The first step is to clean the platform to remove impurities from theplatform surface. The cleaning procedure can be achieved by a number ofmeans, for example by means of an ultraviolet cleaner, by plasmacleaning, or by chemical cleaning using materials such as acids, bases,solvents, gases and liquids.

Once the platform has been cleaned the next step is to apply to thesurface of the metal oxide layer a layer of an adhesion promoting agent.This layer is applied to the platform since capture elements which areto be deposited on the platform might not readily adhere to the metaloxide layer itself. There are several ways in which this layer can beformed. One way is form a layer of a network of silane molecules andanother way is to use what are known as self-assembled monolayers (SAM).These are known techniques which will be apparent to the person skilledin the art. Silanisation for example which can involve a liquid or gasphase is described in Colloids and Interface Science 6, L Boksanyi, OLiardon, E Kovats, 1976, 95-237. The formation of self-assembledmonolayers is described for example in “Ultra thin organic films” byAbraham Ulman, 1991, Academic Press inc. In addition, there are furthermethods available for the immobilisation of capture elements such as

chemical modification of the chip surface with reactive groups and ofthe capture molecules with appropriate linkers (U. Maskos and E. M.Southern, Nucleic Acids Research 1992, vol. 20, 1679-84)

modification of surface and capture molecules with photoreactivelinkers/groups (WO 98/27430 and WO 91/16425)

Immobilisation via coulombic interaction (FP 0 472 990 A2)

coupling via tags (for instance proteine-tag, HIS-tag) in chelatingreactions

and various further methods, for instance as described in Methods inEnzymology Academic Press, New York, Klaus Mosbacher (ed.), Vol. 137,Immobilised enzymes and Cells, 1988.

Plasma induced immobilization/generation of adhesion promoting layerscontaining functional/reactive groups, which enable direct coupling ofcapture molecules or derivatized capture molecules, or indirect couplingof capture molecules or derivative capture molecules via chemicallinkers or photochemical linkers.

An adhesion promoting layer can for example be produced by silanizationwith 3-(glycidoxypropyl)trimethoxysilane (GOPTS). Compounds containingnucleophilic groups such as amines can react with the epoxy function ofthe silane in order to be covalently immobilized. Such a silanizationcan therefore e.g. be used for immobilization of antibodies whichcontain multiple amino groups since antibodies consist of amino acids.In addition, DNA/RNA/PNA strands as capture molecules can also bemodified with amino groups in order to attach these capture moleculescovalently at the platform, as shown in application Example 4 (SNPdiscrimination). In this example, oligonucleotides with amino functionhave been covalently immobilized at the surface of the platform.However, other types of capture molecules can be modified for thispurpose.

In addition, an adhesion promoting layer can be further chemicallymodified in order to alter the surface properties. For example, aGOPTS-silanized platform can be reacted with functionalized saturated orunsaturated organic/hetero-organic/inorganic molecules/derivatives inorder to manipulate hydrophobic/hydrophilic balance of the platform,i.e. change the contact angle of the platform. Furthermore, ionic orpotentially ionic compounds can be used to create positive or negativecharges at the surface of the platform. Capture molecules can be boundeither covalently or by physisorption or by coulombic interaction ofcharged molecules or by a mixture thereof to such modifiedsurfaces/platforms. This is demonstrated in application Example 2 below,where 3-amino-1-propanol is used to modify the surface characteristicsof the GOPTS-silanized platform in a second reaction step in order toimmobilize DNA/RNA/PNA capture molecules. In this example the nitrogen(amine group) introduced at the surface of the platform is quaternizedby protons and provides therefore positive charges which interact withnegative charges of the DNA (polyelectrolyte nature). Instead of3-amino-1-propanol also other organic derivatives of amines, e.g.aliphatic amines, or branched aliphatic amines, or amines containingaromatic or non-aromatic cyclic structures, or amines containinghetero-atoms, or amines containing functional groups, or aminescontaining combinations thereof can be used for the immobilization ofcapture molecules, e.g. DNA/RNA/PNA strands.

Functionalized organic molecules can be used which provide hydrocarbonchains to render the platform more hydrophobic, polar groups can be usedto render the platform more hydrophilic, or ionic groups, or potentiallyionic groups can be used to introduce charges. For instancePolyethyleneglycol (PEG) or derivatives thereof can be used to renderthe platform hydrophilic, which prevents non-specific absorption ofproteins to the platform/surface.

Reactive or photoreactive groups may be attached to the surface of theplatform which may serve as anchor groups for further reaction steps.

A SAM as adhesion promoting layer suitable for immobilization ofantibodies can be obtained by treatment of the platform with amphiphilicalkylphosphates (e.g. stearyl phosphate). The phosphate headgroup reactswith the hydroxy groups at the surface of the platform and leads to theformation of an ordered monolayer of the amphiphilic alkylphoshates. Thehydrophophic alkyl chains render the surface of the platform hydrophobicand thus enable the physisorption of antibodies, as shown in applicationExample 6 (multiplexed immunoassays).

A SAM may also be used for the immobilization of other capturemolecules, e.g. for DNA/RNA/PNA strands. In this case, amphiphilicphosphates/phosphates modified e.g. with amine groups or epoxy groupscan also be used. The capture molecules can be either coupled directlyto the SAM, e.g. to an amine-modified SAM, or after the platform hasbeen reacted with organic derivatives of amines, e.g. aliphatic amines,or branched aliphatic amines, or amines containing aromatic ornon-aromatic cyclic structures, or amines containing hetero-atoms, oramines containing functional groups, or amines containing combinationsthereof, or any other organic, hetero-organic, and/or inorganicmolecules (e.g. epoxy modified SAM).

An adhesion promoting layer may consist of multiple layers in order tomanipulate surface characteristics, e.g. hydrophobicity, contact angle,charge density. In addition, a layer attached to the platform with anyof above mentioned methods may provide or introduce chemicalfunctionality with is required either for the next, subsequent layer, orfor the coupling of capture molecules or derivatized capture molecules.An attachment of chemical linker molecules or photochemical linkermolecules can also be seen as an intermediate layer which enables theattachment of capture molecules to the platform.

This controlled combination of layers/molecules with differentfunctionalities in general is attributed as Supramolecular Chemistry(J-M Lehn, Supramolecular chemistry—Scope and perspectives. Molecules,supermolecules, and molecular devices, (Nobel Lecture, 8.12.1987),Angew. Chem. Int. Ed. Engl., 27, 89, 1988.). The obtained supramolecularstructure provides a functionality which differs from the functionalityof the individual molecules used for the individual layers. For thepresent invention, an intermediate layer can also introduce luminophorsinto such a layer system, which can either be used as energy donor orenergy acceptor/quencher in the sense of Förster Energy Transfer (FRET)or photoinduced electron transfer, or potential sensitive luminophors,before capture molecules or modified capture molecules are attached tothe platform.

For the above-described methods of surface treatment, the followingorganic or inorganic molecules and derivatives thereof can be used:amines, modified amines, jeffamines, aliphatic amines, alcohols, acids,aldehydes, ketones, amides, anhydrides, phosphates, phosphonates,sulfates, sulfonates, thiols, hetero-atom containing compounds, aromaticand aliphatic organic functionalized molecules, aromatic and aliphatichetero-organic molecules,

natural and artificial polymers, silanes, molecules modified withchemical or photochemical active groups, derivatives thereof andfunctionalized, e.g. omega-functionalized derivatives of the listedspecies.

In principle, for the build-up of layer structures consisting of one ormultiple layers, chemical reactive groups and/or chemical groups havingspecial physical or electro chemical properties (e.g. charges) arerequired for the used molecules with all of the above described surfacetreatments.

Either chemical/photochemical interactions (e.g. addition,nucleophilic/electrophilic substitution, radical reaction, condensation,reactions with organic/hetero-organic/inorganic carbonyl derivatives, orphoto-induced reactions, or thermo-induced reactions, Lewis acid/baseconcept), and/or

physical/electrochemical interaction (e.g. Coulomb-interaction,hydrophobic/hydrophilic interaction), and/or

biologic interaction (e.g. antigene/antibody, hybidization,Streptavidin/Avidin-Biotine interaction, agonist/antagonistinteraction), and/or

photochemical/photophysical interaction

may be employed for coupling between molecules/components incorporatedinto such a layer system/adhesion promoting layer.

Adhesion promotion can also be achieved by deposition of microporouslayers or gels on the surface of the platform, the surfacecharacteristics/functionality of the microporous layers or gelsfacilitating deposition of capture elements shortening the requiredincubation time and enhancing sensitivity of the subsequent measures.The microporous layers can comprises organic compounds such as polymers,monomers, molecular assemblies and supra molecular assemblies or it cancomprise inorganic compounds such as glass, quartz, ceramic, silicon andsemiconductors.

An adhesion-promoting layer may be produced by silanisation e.g. using3-(glycidoxypropyl)trimethoxysilane (GOPTS). The adhesion promotinglayer may be further chemically modified in order to alter the surfaceproperties. For example, a GOPTS-silanized platform may be reacted withfunctionalized saturated or unsaturated organic molecules in order tomanipulate the hydrophobic/hydroplilic balance of the platform, andthereby altering the contact angle of the platform.

Once the adhesion promoting layer has been formed on the platform anadditional cleaning step or steps may be necessary to remove excesschemicals used in the preparation of such a layer. After that cleaningthe platform is then ready to receive capture elements.

A two dimensional array of capture or recognition elements is formed onthe 3-D surface of the adhesion promoting layer previously deposited onthe platform. The array of capture elements can be deposited in avariety of ways. Techniques which can be used to deposit captureelements include ink jet printers which have piezoelectric actuators,electromagnetic actuators, pressure/solenoid valve actuators or otherforce transducers; bubble jet printers which make use of thermoelectricactuators; or laser actuators; ring-pin printers; pin tool-spotters;on-chip-synthesis such as that described in WO90/03382 or WO92/10092;very large scale immobilised polymer synthesis (VLSIPS) such as thatdescribed in WO98/27430; photoactivation/photodeprotection of specialdesign photoreactive groups anchored at the surface of the adhesionpromoting layer; microcontact printing; microcontact writing pens;drawing pen or pad transfer/stamping of capture elements; microfluidicschannels and flowcells made by casting from polymer such as PMMA mastersfor example using PDMS (polydimethoxysilane) or by micromechanical ormechanical means, or made by etching techniques for local delivery ofcapture elements; structuring of capture elements by photoablation; ordeposition of capture elements onto gel pads using one of the previouslymentioned techniques or any other photoimmobilisation technique.

The capture or recognition elements which can be deposited onto theplatform are many and varied. Generally speaking the capture moleculesused should be capable of affinity reactions. Examples of recognition orcapture molecules which can be used with the present platform are asfollows:

-   -   nucleotides, oligonucleotides (and chemical derivatives thereof)    -   DNA (double strand or single strand)        -   a) linear (and chemical derivatives thereof)        -   b) circular (e.g. plasmids, cosmids, BACs, ACs)    -   total RNA, messenger RNA, cRNA, mitochondrial RNA, artificial        RNA, aptamers    -   PNA (peptide nucleic acids)    -   Polyclonal, Monoclonal, recombinant, engineered antibodies,        antigenes, haptens, antibody FAB subunits (modified if        necessary)    -   proteins, modified proteins, enzymes, enzyme cofactors or        inhibitors, protein complexes, lectines, Histidine labelled        proteins, chelators for Histidine-tag components (HIS-tag),        tagged proteins, artificial antibodies, molecular imprints,        plastibodies membrane receptors, whole cells, cell fragments and        cellular substructures, synapses, agonists/antagonists, cells,        cell organelles, e.g. microsomes small molecules such as        benzodiazapines,    -   prostaglandins,    -   antibiotics, drugs, metabolites, drug metabolites    -   natural products    -   carbohydrates and derivatives    -   natural and artificial ligands    -   steroids, hormones    -   peptides    -   native or artificial polymers    -   molecular probes    -   natural and artificial receptors    -   and chemical derivatives thereof    -   chelating reagents, crown ether, ligands, supramolecular        assemblies    -   indicators (pH, potential, membrane potential, redox potential)    -   tissue samples (tissue micro arrays)

The activity or density of the capture molecules can be optimised in anumber of ways. The platform with the capture elements deposited thereoncan be incubated in saturated water vapour atmosphere for a definedperiod in order to rehydrate the printed loci. This optimises thedensity of the capture molecules, i.e. increases available binding sitesper unit area Subsequently the incubated chips can be baked for adefined period, say 1 minute at 80° C. for cDNA capture molecules. Theplatform can be washed by wetting with a small amount of pure water orany other suitable liquids or solutions to avoid cross contamination ofthe capture elements by excess unbound material. After these procedures,the prepared platform can be stored in a dessicator until use. Prior touse of the chip, an additional washing procedure with 0.1 to 10 mlhybridization buffer or other suitable solutions/liquids may be requiredto reactivate/rehydrate the dried capture elements and to further removeexcess unbound capture elements/buffer residues. In the case of DNAcapture molecules, the washing procedure has found to be most effectivewhen performed at a temperature between 50 and 85° C.

Process steps for the chip handling can be automated by usinghybridization stations such as e.g. the GeneTAC Hybridization stationfrom Genomic Solutions Inc., Michigan, US.

The particular measurement technique to be described is that involvingluminescence in particular fluorescence. In carrying out a measurement,a sample to be investigated is placed on the sensing area of theplatform on which the capture elements have been provided. In order toachieve fluorescence, fluorophores are added to the system prior to themeasurement being taken. The fluorophores can be added to the sample forexample as labelled affinity partners although it is also possible toattach fluorophores to the capture elements on the platform. Themeasurements are based upon the fact that fluorescent emission from thecapture elements containing labelled capture molecules and/or fromlabelled affinity partners is altered by its interaction with theanalyte or sample under investigation. Labels of different excitationand emission wavelength can be used, there being one or severaldifferent labels, label 1 being for a control experiment and label 2 forthe experiment.

FIG. 3 shows schematically the energy profile of the evanescent field atresonance position and how it extends beyond the surface of the metaloxide layer (32) so that it can excite fluorophores in the closevicinity of the surface of the sensing area, e.g. fluorophores attachedto capture molecules or fluorophores attached to molecules bound to thecapture molecules (38). The evanescent field decreases exponentially tozero within approximately one micron.

It will be appreciated that in carrying out an analysis one or multiplemeasurements are made. One can be background measurement prior to thesample being brought into contact with the capture elements. A secondmeasurement can be made with/after the sample has been brought incontact with the capture elements. For comparison of multiple samples,for instance “control” and “treated” sample in gene expressionexperiments, the chip can be regenerated after the “control” experimentas described in the application example 2, and a further backgroundmeasurement and a measurement after/with the “treated” sample (was)applied to the chip can be registered. To gain information regarding thereaction kinetics of the affinity partners, a complete set ofmeasurements can be recorded as a function of incubation time and/orpost-wash time. A typical arrangement for such a measurement is shown inFIG. 6. The platform shown in FIG. 2 is adjusted to the angle at whichevanescent resonance is achieved and a measurement of the fluorescenceemitted from the surface of the platform is made using the CCD camera(66). This provides an indication of the fluorescence emitted from eachposition on the array of capture elements deposited on the platform.This can be analysed to deduce the affinity of the reactions which haveoccurred between the capture elements and the sample underinvestigation.

An arrangement as shown in FIG. 6 captures the whole luminescence, e.g.fluorescence, image of the entire platform at one shot without the needof any moving parts during measurement. Such a non-scanning device canbe very simple and cheap and is especially suited for point-of-careapplication or portable systems. Another typical arrangement confinesthe coherent laser light down to micrometer dimensions by means ofoptical elements thereby increasing the electrical field in the focalpoint and scans the sensing area or areas.

It will be appreciated that a wide variety of samples can be analysedusing the present technique. The sample is generally taken to be theentire solution to be analysed and this may comprise one or manysubstances to be detected. The sample may be a solution of purified andprocessed tissue, or other materials obtained from biopsy andexamination investigation research and development including sample fordiagnostic purposes. The sample may also be a biological medium such asegg yolk body fluids or components thereof, such as blood, serum andurine. It may also be surface water, solution or extracts from naturalor synthetic media, such as soils or parts of plants, liquors frombiological processes or synthetic liquors.

In order to carry out the measurement, the sample may be introduced intoa sample cell of the type shown in FIGS. 4 a and 4 b of the drawings.This cell comprises a housing (41) which is made from a polymer such asPMMA This polymer has been machined to define a central compartment (44)with dimensions corresponding to the dimensions of the platform. Afurther depression is formed in the compartment (44) to define a chamber(46) which is sealed around its edge by an O-ring (47). The chamber (46)is open at its top and bottom. Solution to be analysed can be introducedinto the chamber (46) within the O-ring (47) through a flow line (45).Flow within the flow line (45) can be controlled by a valves (43). Thecell includes a cover (49) which can be located over and secured tohousing (41) to close the top of the cell. The cover (49) includes awindow (50) which locates over the compartment (46) and thereby allowsradiation to pass through the cover and into the cell (46).

In use of the cell, the housing (41) is located against the surface ofthe platform which has the capture elements formed thereon so that thelid (49) is remote from that surface. This brings the compartment (46)into communication with the sensing area of the platform. The sample tobe investigated is then fed into the compartment (46) through the flowline (45) so that it is brought into contact with the capture elementson the surface of the platform. A measurement of the fluorescenceinduced at various capture points is then carried out as previouslydescribed.

FIG. 8 illustrates possible alternative forms of the platform.

The sensing elements can be arranged in various ways, for instancerectangular, circular, hexagonal-centric, elipsoidal, linear orlabyrinthine. The sensing area may be rectangular, round or of any othershape. The grooves may be arranged either equidistant linear orequidistant circular, or may correspond to segments of such structures.

The platform can be either rectangular or disc-shaped, or of any othergeometry. The platform can comprise one or multiple sensing areas, eachsensing area can comprise one or multiple capture elements, and eachcapture element can comprise one or multiple labelled or unlabelledcapture molecules.

The platform can also be adapted to microtiter-type plates/devices inorder to perform one or multiple assays in the individual microtiterwells. This can be achieved for all plate types: 96, 384, 1536, orhigher numbers of wells, independently of the dimensions of therespective microtiter-plate.

The following is a specific example of a platform:

1. Physical Performance of 3-D Platform: Abnormal Reflection

1a. Platform 1

The gene chip transducer platform comprises a planar, transparentsubstrate (glass AF45 by Schott) of 0.7 mm thickness. Into the substratea periodic surface structure is etched by photolithographical means(deposition of photoresist, <1 μm; writing of periodic structure intoresist either by two beam interferometry/holography; etching the resistwith reactive ion etching using Ar gas; stripping of residualphotoresist).

The shapes of the surface structure are close to sinusoidal. The width(period) of a single structure is 360 nm. The depth of the grooves areapproximately 38 nm.

On top of the homogeneously structured glass surface a dielectrictransparent metal oxide film (Ta₂0₅) with high refractive index of app.2.2 at 633 mm wavelength is deposited. The process is by ion plating.The layer thickness is 130 nm. The primary structure/architecture of theglass surface is transferred to the top of the metal oxide layer due tothe highly energetic and anisotropic deposition process.

When a highly parallel, expanded, coherent laser beam is directed ontothe transducer at distinct angles θ, corresponding to a so-calledresonance position, almost all light is reflected by the transducerplatform and the 0. order transmission intensity is reduced to less than1% (compared to 90-95% at an arbitrary angle).

The width Δθ of the resonance condition, where almost all the light isreflected, is proportional to the wavelength λ (633 nm, fixed) and tothe radiation loss coefficient α. The radiation loss coefficient isgoverned by the depth of the grating grooves, geometry and duty cycle ofthe corrugated structure, and increases almost quadratically withincreasing groove depths. For our case (laser wavelength 633 nm, 130 nmmetal oxide layer, 38 nm groove depth) the radiation loss is app.2000/cm, i.e. the propagation distance of a guided laser beam in such alayer system before it is diffracted out of the platform by the periodicstructure is 1/2000 cm,=5 μm. This is a surprisingly short distance.Therefore under these conditions no waveguiding occurs. By refinement ofthe platform specifications the propagation distance can be furtherreduced.

For characterization of the resonance effect, the intensity of theparallel beam (TE polarisation) is adjusted to 100 μW for a 4 mmdiameter area (power meter Newport NRC 1835). The angle between platformnormal and incident beam is rotated 1 to 2 degrees away from the centreposition of the anomalous reflection (resonance condition). The centreposition is at 2.5°. The platform is then rotated in steps of 5/1000°(Newport NRC controller PM 500) and the change of the power of thetransmitted beam monitored. At resonance angle, less than 1% (<1 μW) ofthe original transmitted beam reaches the detector. The power of theincident laser beam is reflected totally (specularly reflected beam:approximately 100%).

The full width at half maximum of the resonance for abnormal reflection(FWHM) is in our case 0.9°. The homogeneity of the reflection over thewhole transducer surface (18 mm×18 mm) is better than 90%.

1b. Platform 2

The shapes of the surface structure are close to rectangular. The width(period) of a single structure is 360 mm. The depth of the grooves areapproximately 52 nm. On top of the homogenously structured glass surfacea dielectric transparent metal oxide film (Ta205) with high refractiveindex of approximately 2.15 at 633 nm wavelength is deposited. Theprocess is by sputtering. The layer thickness is 150 nm. The primarystructure/architecture of the glass surface is transferred to the top ofthe metal oxide layer due to the highly energetic and anisotropicdeposition process.

When a highly parallel, expanded, coherent laser beam is directed ontothe transducer at distinct angles θ corresponding to a resonanceposition, almost all light is reflected by the transducer platform andthe 0. order transmission intensity is reduced to less than 1% (comparedto 90-95% at an arbitrary angle).

The width Δθ of the resonance condition, where almost all the light isreflected, is proportional to the radiation loss coefficient α. Theradiation loss coefficient for our case (laser wavelength 633 nm, 150 nmmetal oxide layer, 52 nm groove depth) is above 2000/cm, i.e. thepropagation distance of light launched into such a layer system beforeit is diffracted out of the platform by the periodic structure is 1/2000cm,=5 μm. Therefore under these conditions no waveguiding occurs.

For the characterization of the resonance effect, the intensity of theparallel beam (TE polarisation) is adjusted to 600 μW for a 4 nundiameter area (power meter Newport NRC 1835). The angle between platformnormal and incident beam is rotated 4 degrees away from normalincidence. The platform is then rotated in steps of 5/1000° (Newport NRCcontroller PM 500) and the change of the power of the transmitted beammonitored. At resonance angle, less than 0.5% (<3 μW) of the originaltransmitted beam reaches the detector. The power of the incident laserbeam is reflected totally (specularly reflected beam approximately100%).

Due to the widening of the resonance width of platform 2 according todeeper grooves compared to platform 1 the resonance curves of the +1 and−1 diffraction order overlap creating a single extremely wide resonancelocated exactly at normal incidence. The full width at half maximum ofthe resonance for abnormal reflection (FWHM) is in our case 4.2°. Thehomogeneity of the reflection over the whole transducer surface (18mm×18 mm) is better than 95%.

2. Example for Gene Expression Analysis

a) Preparation

Sensor platforms (dimensions 18×18 mm²) of the type described withreference to FIG. 2 wee first sonicated twice in chloroform (FLUKA,“purriss.”) and subsequently twice in isopropanol (Merck, “Uvasol”);each for 15 min. The platform were then dried in vacuum and cleaned in aUV cleaner for 30 min (Boeckel industries Inc, model 135500). O-xylenewas heated to 75° C. (stirring) and 2% v/v 3-glycidoxypropyltrimethoxysilane (Fluka, “purum”) as well as 0.2% v/vN-ethyldiisopropylamine (Fluka, “purum”) were added to the heatedsolvent (stirring). The platforms were mounted into racks and thenincubated for 7 h in the solution at 75° C. (stirring). Subsequently,the platforms were sonicated three times in fresh acetonitrile (Fluka,“HPLC grade”), each for 15 min. The platforms were then sonicated in asolution of 2% v/v 3-amino-1-propanol (Fluka, “purum”) in acetonitrilefor 15 min and then incubated in the same solution over night at roomtemperature (stirring). Next day, the platforms were first sonicatedthree times for 15 min in fresh isopropanol (Fluka, “HPLC grade”) andthen three times for 15 min in flesh methanol (Merck, “Uvasol”).Finally, the platforms were dried and stored in vacuum.

b) Immobilization of Recognition Elements

Arrays consisting of 10 different cDNAs (each cDNA 10 replicates: CYP450 1A1, CYP 450 2B1 EST., CYP 450 2B1, CYP 450 2B2, CYP 450 3A1 human,CYP 450 3A2, CYP 450 4A1, β-actin, GAPDH, external standard) wereprinted on the platforms with an ink-jet printer (Microdrop GmbH,Norderstedt, Germany). The concentration of the cDNA solutions was 50ng/μL. The diameter (10 ink-jet droplets/position) was about 250 μm andpitch of the spots was about 500 μm. Therefore, the overall dimension ofthe 10×10 arrays was about 5×5 mm². The layout and assignment of theimmobilized cDNA spots is schematically shown in FIG. 5. The arrays wereprinted in the centre of the platforms with the dimensions (18×18 mm²).Subsequently, the platforms were incubated over night in a closedcontainer in saturated water vapor atmosphere. Next day, the incubatedchips can be baked for a defined period, say 1 minute at 80° C. Then theplatforms were flushed with deionized water and dried with nitrogenflow.

c) Detection Set-Up

The detection set-up used is shown schematically in FIG. 6. Anexcitation laser (61) (HeNe laser, 633 nm, 1.3 mW) and a 20× beamexpander (64) were jointly mounted (62) onto a goniometer (63). Theexpanded laser beam was directed towards the platform (67) by means of adichroic mirror (68). The center of rotation for the laser beam lay inthe plane of the metal oxide layer of the platform (67). Thefluorescence emitted from the platform surface was collected via thedichroic mirror (68). Additional fluorescence filters (65) were used toseparate fluorescence (69) from excitation light. A cooled CCD (AstrocamEEV 30/11) camera (66) equipped with a Nikon Noct lens (NumericalAperture 1.2) was used to measure fluorescence images from the surfaceplatforms. The goniometer allowed the adjustment of the angle of theincident expanded laser beam with respect to the surface normal of theplatform. Fluorescence images were taken under evanescent resonanceconditions (i.e. the incident expanded laser beam was adjusted to thatangle where the light transmitted through the platform shows a minimum).

d) Chip Cartridge

The platform were mounted to the specially designed cartridge (41) madefrom PMMA/polymer which is schematically shown in FIG. 4. The depression(44) had dimensions (18×18×0.7 mm³) and the incubation chamber (46) was0.2 mm deep. The solution in the incubation chamber was exchanged viaflow channels (45) of 0.5 mm diameter that were drilled into the PMMA.The content of the cartridge was be exchanged via the inlet/outlet (42).The platform was positioned in the corresponding depression of thecartridge with the sensing area directed towards the incubation chamber.The cover (49) was fixed to press the platform against the sealing. Themilled/micromachined window (50) in the cover allowed illumination withexcitation light and the acquisition of fluorescence images of theplatform surface. The valve-to-valve-volume of the cartridge was about14 μL.

e) Denaturation Unit

A thermoelectric element was used to control the temperature fordenaturation (79° C.), incubation, (42° C.) regeneration (79° C.) andwash (42° C.) of the platform in the flow cartridge.

f) Sample Preparation

2 groups of rats (each 3 rats) were used for the present study. Onegroup (treated) was treated with 80 mg phenobarbital, sodium salt, insaline (0.9% w/v NaCl) per kg body weight and the second group (control)only with 0.9% NaCl. One daily treatment on 4 consecutive days wasgiven. At the end of the 4 days, the animals were sacrificed and liversamples were snap frozen in liquid nitrogen and stored at 40° C.

Subsequently, the total RNA/mRNA was isolated and labeled by reversetranscription into first strand cDNA (incorporation of labeleddeoxynucleotides, same or different fluorophore labels for control andtreated), purified and dissolved in 20 μL hybridization buffer.

g) Assay Processing

A Cavro stepper syringe was used to pump/aspirate buffers and solutionsinto the cartridge. The following steps were executed to measure the CYP450 induction in rat:

1) 30 min pre-wash at 79° C. with 1 ml hybridization buffer (HB),measurement of background 1 of the platform in contact with HB.

2) Injection of “control” sample into cartridge, 30 min denaturation at79° C., then over night incubation at 42° C.

3) 10 min post-wash at 42° C. with 1 mL HB, measurement of “control”intensities of platform in contact with HB.

4) Regeneration: 30 min wash at 79° C. with 1 mL hybridization buffer(HB), measurement of background 2 of the platform in contact with HB.

5) Injection of “treated” sample into cartridge, 30 min denaturation at79° C., then over night incubation at 42° C.

6) 10 min post-wash at 42° C. with 1 mL HB, measurement of “treated”intensities of platform in contact with HB.

All fluorescence images were measured under evanescent resonanceconditions.

h) Data Processing

The net intensities (control-background 1 and treated-background2) ofall spots were calculated and all intensities of the treated data setwere normalized by the help of the intensities of the external standard.The expression ratios (fold change) between the respective genes werecalculated by division offold change=normalized treated/control.Mean values of the each 10 replicates were calculated.i) ResultsER-Chips measured under evanescent resonance conditions showed ingeneral about 100 fold stronger intensities and improvedsignal/background ratios.

The mean values for fold change are summarized in the following table:GENE Fold change CYP 450 1A1 (rat) 1.6 CYP 450 2B1 EST.(rat) 16 CYP 4502B1 (rat) 25 CYP 450 2B2 (rat) 32 CYP 450 3A1 (human) 3.2 CYP 450 3A2(rat) 2.5 CYP 450 4A1 (rat) 1.6 β-actin (rat) 2.1 GAPDH (rat) 2.33. Example to Illustrate Enhanced Amplification

A platform was prepared and processed according to the example justdescribed. Subsequent to the incubation with sample, 2 images were takenwith the CDD camera detection set-up described above with reference toFIG. 6. The first image was taken in epifluorescence mode withoutadjustment to conditions for evanescent resonance (“epifluorescence” inFIG. 7 a). The second image was taken under evanescent resonanceconditions (“ER enhancement” in FIG. 7 b), i.e. the angle of theincident laser beam was adjusted with respect to the surface normaluntil the light transmitted through the chip showed a minimum. The imageprofiles (net signals) show that intensities measured withER-enhancement are about 100 fold stronger then the intensities obtainedwith conventional epifluorescence.

In the example described above a single sample cell (41) is used tobring the sample into contact with the sensing area of the platform. Itwill be appreciated that microtiter type sample container can be used incompensation with a platform having a plurality of sensing areas toallow measurement of a plurality of samples and thereby improvemeasurement efficiency.

4. Oligonucleotide Microchip for Discrimination of Single NucleotidePolymorphism (SNP)

a. Chip Preparation

Sensor platforms (dimensions 18×18 mm2) of the type described withreference to FIG. 2 we first sonicated twice in chlororform (FLUKA,“purum”) and subsequently twice in isopropanol (Merck, “Uvasol”), each15 min. The platforms were then dried in vacuum and cleaned in a UVcleaner for 30 min (Boeckel Industries Inc, model 135500). O-xylene washeated to 75° C. (stirring) and 2% v/v3-glycidoxypropyl-trimethoxysilane (Fluka, “purum”) as well as 0.2% v/vN-ethyldiisopropylamine Fluka, “purum”) were added to the heated solvent(stirring). The platforms were then mounted into racks and thenincubated for 7 h in the solution at 75° C. (stirring). Subsequently,the platforms were washed three times with fresh acetonitrile (Fluka,“HPLC grade”) each for 15 min. Finally the platforms were dried andstored under vacuum.

b. Immobilization of Capture Elements

Two different amino-modified oligonucleotides (capture probes) wereprinted in a checkerboard-like layout (5×5=25 spots) onto the silanizedplatforms. A GMS 417 ring-pin arrayer was used for the printing (GeneticMicrosystems, Boston, Mass.). The concentration of the oligonucleotidesused as capture molecules was 100 nmol/ml. Diameter of the spots was 125micrometer with 500 micrometer centre-to-centre distance. The 2oligonucleotides were called cPM (“capture perfect match”) and cMM(“capture mismatch”) and differ only in one base: PM:Cy5-5‘GTGTTAAGGTGT3‘ MM: Cy5-5‘GTGTTGAGGTGT3‘cPM and cMM were labeled with an amino group at the 5′ end which enablescovalent binding of the oligonucleotides to the epoxy-functionalizedplatforms. The arrays were printed in the centre of the platforms withthe dimensions (18×18 mm2). Subsequently the platforms were incubatedover night in a closed container in saturated water vapour atmosphere.Next day the chips were dried and the washed with 1 ml of aqueous 50%urea solution Alternatively, an aqueous bovine serum albumine solution(BSA, 1 mg/ml) was used. After blocking the chips were flushed withdeionized water and then dried with nitrogen flow.c. Detection Set-UpThe CCD set-up described in the previous example was used.d. Chip CartridgeThe cartridge described in the previous example was used.e. Analytes/Samples

Two Cy5-labeled oligonucleotides called PM (“perfect match”) and MM(“mismatch”) with sequences complimentary to the immobilized captureoligonucleotides cPM and cMM were used as the analytes: cPM:3‘CACATTCCACA5‘-NH₂ cMM: 3‘CACAACTCCACA5‘-NH₂The concentration of the analyte solutions was 30 pM each.f. Assay ProcessingA platform was first washed 2 times with 1 ml of hybridization buffer(HB), with 1 min delay between the washings. Subsequently about 15microliter PM analyte solution (30 pM) was injected into the flowcartridge. After 30 min of incubation, the platform was washed with 1 mlof HB and a fluorescence image (“PM” in FIG. 9 a) was taken in theresonance position of the chip. Subsequently, the bound fluorescencelabeled oligonucleotides were removed (stripped) by injection of 2×1 mlof an aqueous 50% urea solution with 2 min of delay between theinjections. After additional 2 min the chip was washed by injection of2×1 ml HB with 2 min delay between the injections and a fluorescenceimage (“regeneration” in FIG. 9 b) was taken in the resonance positionof the chip. Finally, approximately 15 microliter MM analyte solution(30 pM) was injected into the flow cartridge. After 30 min ofincubation, the platform was again washed with 1 ml of 1B and afluorescence image (“MM” in FIG. 9 c) was taken in the resonanceposition of the chip. All steps were performed at room temperature.g. Data Processing

The mean intensity of the 2 different groups of capture spots (cPM andcMM) was calculated for both experiments (incubation of 30 pM PM analyteand 30 pM MM analyte). In addition, the difference of the mean intensitycPM-cMM and the ratio cPM/cMM was calculated.

h. Results

All data calculated were summarized in the following Table. The twooligonucleotide analytes, Cy-5 labelled PM and Cy-5 labelled MM, whichdiffer only in one base (SNP) can be clearly distinguished from theobtained data. 30 pM PM analyte 30 pM MM analyte mean intensity meanintensity [counts] [counts] cPM spots 1140 704 cMM spots 24 2075 cPM −cMM 1116 −1371 cPM/cMM 48 0.345. Example for Antibody Immuno AssayPrimary antibodies are spatially resolved immobilized (for instancecheckerboard pattern) on the surface of the sensor platform. The bindingof the antigens to be detected and of the luminescence-labeled secondaryantibodies (used for the detection of a second epitope of the individualantigens to be detected) is achieved by subsequent incubation, firstwith the analyte containing the various antigens in differentconcentrations, and then with the luminescence-labeled secondaryantibodies.

Alternatively, the antigenes (analyte) and luminescence-labeledsecondary antibodies can be mixed in a pre-incubation step, which allowscomplexation of luminescence-labeled secondary antibodies with theantigenes. After this pre-incubation step, the sensor platform surfaceis incubated with the mixture.

The luminescence labeled immuno complexes bound to the surface werequantified with the ER-set-up. (Pre-wash with suitable buffer, PBS, andpost-wash if required).

6. Protein-Microchips for Multiplexed Immuno Assays

a. Chip Preparation

Sensor platforms (dimensions 18×18 mm2) of the type described above weresonicated twice in chlororform (FLUKA, “purriss.”) and subsequentlytwice in isopropanol (Merck “Uvasol”), each 15 ml. The platforms weredried in vacuum and cleaned in a UV cleaner for 30 min (BoeckelIndustries Inc, model 135500). The chips were placed in a smallcontainer and stored in a 0.5 mMolar solution of octadecylphosphate inpropanol for 24 hours. Subsequently, the chips were washed with 5 mlisopropanol in order to remove excess allylphospate and dried in anitrogen flow. This procedure created a Self-Assembled-Monolayer (SAM)of alkylphosphate at the surface of the platforms. This adhesionpromoting layer rendered the platform hydrophobic (contact angle about100°) and enabled the adsorption of proteins on the platform byhydrophobic interaction

b. Immobilization of Capture Elements

Two different monoclonal antibodies, anti-human Chorinonic Gonadotropin(anti-hCG) and anti-Interleukin 6 (anti-IL6) were printed in acheckerboard-like layout onto the hydrophobic platforms in saturatedwater vapour atmosphere (4×4 array, 8 spots for each antibody). Theconcentration of the capture antibody solutions was 400 and 100microgramm/ml respectively. An ink-jet-printer was used for the printing(Microdrop, Norderstedt, Germany). The diameter of the spots was 150micrometer with 320 micrometer centre-to-centre distance. The printedarrays were incubated for 2 hours in a closed container in saturatedwater vapour atmosphere. Subsequently, the chips were dried and flushedwith 10 ml phosphate buffered saline (PBS) solution containing 10% obovine serum albumin (BSA), 5% sucrose and 0.02% sodium azide. Thiswashing step blocked the hydrophobic surface of the chips by adsorptionof BSA and rendered the surface more hydrophilic after the captureelements had been immobilized. As a consequence, the blocking procureprevented non-specific binding of proteins to the platform which couldcause increased background fluorescence. After blocking, the chips wereflushed with deionized water and dried with nitrogen flow. The platformswere stored in a refrigerator until use.

c. Detection Set-Up

The CCD set-up described in Example 2 was used.

d. Chip Cartridge

The cartridge described in Example 2 was used.

e. Analytes/Samples

3 analyte solutions were prepared:

-   I) a solution coning 500 ng/ml Cy-5 labelled IL6, antigene-   II) a solution containing 50 ng/ml Cy-5 labelled human Chorionic    Gonadotropin (hCG), antigene-   III) a preincubated mixture (1 hour) containing 50 ng/ml IL6 and 100    ng/ml polyclonal anti-IL6 antibody labelled with Cy5.    PBS pH 7.0 containing 1% BSA is used as solvent for the analytes.    f. Assay Processing    Three platforms (as described under 6b.) were prepared for the    analyte incubation (cf. e.) by mounting the platforms into    cartridges and washing with 1 ml of PBS pH 7.0 containing 1% BSA.    Subsequently, about 15 microliter of the analyte solutions I), II)    and III) were injected into the flow cartridges. The incubation time    was 2 hours each for 1) and II). For analyte III) the incubation    time was 12 hours. After incubation of the analyte, the platforms    were washed with 1 ml of PBS pH 7.0 containing 1% BSA. Fluorescence    images were taken from the chips under off-resonance conditions (epi    fluorescence, about 7° apart from resonance angle) and under    resonance condition for each chip (resonance, incident light about    2.5° with respect to normal). Images and data obtained are shown in    FIG. 10.    All steps were performed at room temperature.    g. Data Processing    The mean intensities and the background of the spots were calculated    by help of Imagene Array software (Biodiscovery, Los Angeles,    Calif.). Spot mean in FIG. 10 represents the background corrected    mean intensity of the respective capture elements under interest. In    addition, the noise was calculated as standard deviation of the    background from the fluorescence images. Therefore, signal/noise in    FIG. 10 corresponds the ratio of spot mean over background standard    deviation.    h. Results    All data calculated are summarized in FIG. 10. Images and data    obtained in epifluorescence mode as well as under resonance    conditions are summarised in FIG. 10. Rows I) and II) show the    results of an immunoreaction between immobilized monoclonal capture    antibodies and labeled antigenes (Cy5-labelled IL6 and hCG    respectively).    Row III) corresponds to a sandwich type immuno reaction between an    immobilized monoclonal antibody (anti-IL6) and a preincubated    mixture of IL6 antigene and a CY5-labelled secondary polyclonal    antibody against IL6.    For the results of row I) the signal intensity increases from 46    counts in epi fluorescence mode to 1100 counts in resonance mode of    the platform. This corresponds to an enhancement factor of about 24    regarding the spot mean values. The signal/noise ratio improves from    7.0 (epi fluorescence) to 69.2 (resonance), corresponding to a    factor of 10.    For the results of row II) the signal intensity increases from 32    counts in epi fluorescence mode to 646 counts in resonance mode of    the platform. This corresponds to an enhancement factor of about 20    regarding the spot mean values. The signal/noise ratio improves from    5.0 (epi fluorescence) to 75.1 (resonance), corresponding to a    factor of 15.    For the results of row III) the signal intensity increases from 25    counts in epi fluorescence mode to 296 counts in resonance mode of    the platform. This corresponds to an enhancement factor of about 12    regarding the spot mean values. The signal/noise ratio improves from    3.8 (epi fluorescence) to 44.1 (resonance), corresponding to a    factor of 12.    Spot mean and signal/noise values for all 3 assays are at least one    order of magnitude higher for the chips in resonance mode compared    to the same chips in non-resonance mode (epi fluorescence). The    fluorescence images of rows I) and II) are complementary    (checkerboard-layout). All chips used have the same set of capture    elements, i.e. monclonal anti-hCG and monoclonal anti-IL6.    Examples 7a, 7b, 8 and 9 Describe the Additional Amplification    Obtained when Using TM Excitation and the Enhanced Signal Intensity    when Directing the Incident Beam onto the Transparent Substrate Side    of the Platform (Chip)

Physical properties of the platforms (Chips 1 to 6) used in Examples 7a,7b, 8 and 9 are as follows. Groove depth means depth of corrugationlayer. The high-refractive index layer is pure Ta₂O₅. The angles of therespective resonance positions are with respect to normal incidence(0°). Groove Thickness Resonance Resonance Example depth of Ta₂O₅ angleTE angle TM 7a) chip 1 55 nm 168 nm 0.8°   9° 7a) chip 2 40 nm 161 nm0.8°   9° 7b) chip 3 35 nm 180 nm 4.25°  3.15°  8) chip 4 35 nm 150 nm  2° 7.7° 9) chip 5 40 nm 185 nm 6.5° 2.0° 9) chip 6 55 nm 190 nm 6.0°2.6°7a) Comparison Between TE and TM Polarized Excitation Light for a CCDSet-Up with Dichroic Mirror.The experimental set-up of FIG. 6 is used for each measurement. Thehardware is the same for both parts of the experiments. The platform(chip) is oriented in a way that the grooves are perpendicular to theplane of drawing FIG. 6.TE part: The laser beam is adjusted so that the expanded beam strikeschip 1 at normal incidence with the incident light polarised so as toprovide TE excitation. The laser is rotated on a goniometer within theplane of the drawing until the angle of abnormal reflection is reached.The fluorescence signals of a variety of spots excited by the laserlight are recorded by the CCD camera. This is repeated using chip 2.TM part: the incident laser light is rotated by 90 degrees and the beamadjusted so that the expanded beam strikes chip 1 at normal incidence.This geometry creates TM excitation. The laser is rotated on agoniometer within the plane of the drawing until the angle of abnormalreflection is reached. The fluorescence signals of a variety of spotsexcited by the laser light are recorded by the CCD camera This isrepeated using chip 2.Resulting peak intensities at the abnormal reflection angle are asfollows:TE/TM comparison: For chip 1 the maximum of the fluorescence signal forTE excitation reaches 2,750 counts, for TM almost 9,000 counts. For chip2 the values are 10,000 vs. 40,000 counts respectively.7b) Example with CCD Camera to Further Demonstrate Amplification by TMExcitationUsing chip 3, the platform according to FIG. 6 is aligned so that theplane of polarization of the linearly polarize and expanded laser lightis perpendicular to the longitudinal axis of the grooves of thecorrugated surface, i.e. giving rise to TM excitation. Using thisgeometry and orientation, the fluorescence yield is even higher whencompared to the arrangement where the plane of polarization is parallelto the grooves (TE excitation).The angle of the light incident on the chip is varied between 0° (normalincidence) and 10° with respect to the surface normal. The maximum ofthe fluorescence intensity is found at 3.15°. Fluorecence images aretaken by means of the CCD camera as a function of the angle of the lightincident on the chip. The mean value of the fluorescence intensity of 32selected spots is calculated and the mean background intensity issubtracted for every image.Obtained fluorescence intensities, background values, and the relativechange of the fluorescence as function of the angle of the incidentlight are summarized in the table below.

For the net fluorescence signal (signal minus background) we measure anenhancement of fluorescence yield by a factor of 223. Signal BackgroundSignal − background Normalized Angle [°] (counts) (counts) (counts)signal 0 28.7 23.6 5.1 1 1 30.4 22.9 7.5 1.5 2 36.3 24.1 12.2 2.4 3203.8 26.4 177.4 34.8 3.1 723.9 29.0 694.9 136.3 3.125 979.6 29.7 949.9186.3 3.15 1170.9 30.7 1140.2 223.6 3.175 1132.0 30.3 1101.7 216.0 3.2955.9 31.9 924.0 181.2 3.225 523.8 27.1 496.7 97.5 3.5 73.7 29.4 44.38.7 4 40.5 26.5 14.0 2.8 7 30.4 25.3 5.1 1 10 30.0 23.8 6.2 1.28. Comparison Between TE and TM Polarized Excitation Light for a CCDSet-Up with Dichroic MirrorThe experimental set-up differs from that in Example 7: the detector isrotated in the horizontal plane around chip 4 which is excited atresonance position. The detector measures the fluorescence emitted intothe whole solid angle. The results are illustrated in FIGS. 11 and 12.With excitation of the TM configuration the measured fluorescenceintensity exceeds the saturation level of 4096 (12 bit camera), whereaswith TE configuration intensities are up to 400 counts.Baseline/background for both experiments is approx. 75 counts.9. Example with Laser Scanner to Demonstrate Amplification by TE vs. TMOrientation, with Results on Incident Light onto the High RefractiveIndex Side Compared with Light Incident onto the Substrate SideA laser scanner (GSI Lumonics ScanArray 4000) is used to obtain thefluorescence images. The laser scanner focusses the excitation laserlight (632.8 nm) to a diameter of 10 μm and a photomultiplier collectsthe fluorescence photons at a given position of the platform (hereafter:chip). A 2 dimensional image is created by scanning across the chip in10 μm steps. The fluorescence intensities of each chip are measured at 4different orientations:9a) chips oriented with laser light incident from the corrugated highrefractive index side (abbreviated CHRIS); polarisation parallel tolongitudinal axis of grooves (“TE”;9b) chips oriented with laser light incident from the corrugated highrefractive index side; polarisation perpendicular to longitudinal axisof grooves (“TM”);9c) chips oriented with laser light incident from substrate side;polarization parallel to longitudinal axis of grooves (TE);9d) chips oriented with laser light incident from substrate side;polarization perpendicular to longitudinal axis of grooves (TM).

The two individual chips 5 and 6 are analyzed. The mean value of thefluorescence intensities of 8 selected spots is calculated for each chipand the results are given in the table below: Fluores- Light cenceNormal- incident signal ized Chip Orientation of polarisation from(counts) value Chip 5 parallel to grooves (TE) CHRIS 979 1 Chip 5parallel to grooves (TE) substrate 7059 7.2 Chip 5 perpendicular togrooves (TM) CHRIS 6661 6.8 Chip 5 perpendicular to grooves (TM)substrate 42669 43.6 Chip 6 parallel to grooves (TE) CHRIS 1077 1 Chip 6parallel to grooves (TE) substrate 5116 4.8 Chip 6 perpendicular togrooves (TM) CHRIS 7415 6.9 Chip 6 perpendicular to grooves (TM)substrate 52826 49.0

It will be appreciated that many alternatives to the describedembodiments are possible. Thus different excitation geometries may beexploited depending on application area, requirements, costs,geometrical restrictions etc.

Another feature of the present platform is that it allows larger sets ofdata to be acquired in parallel. Also it can be used several times.Immobilised affinity complexes can be regenerated at elevatedtemperature using organic solvents and/or chaotropic reagents (saltsolutions) while maintaining the binding capacity substantiallycompletely.

In the description given above the whole area of a sensing region isirradiated. It is possible also to use a laser with a non-expandedfocused beam and to scan the sensing area so that end capture element isexcited in turn. This arrangement permits the use of a cheaperphotodetector than the CCD camera e.g. a photomultiplier, or avalanchephotodiode can be used. Also this arrangement will enhance fierier thesensitivity due to the fact that laser energy is more confined.

It is possible also to design platforms in accordance with the presentinvention for use as microscope slides thereby allowing them to be usedwith a fluorescence microscope.

The platforms can also be designed for use with large scale microfluidicsystems such as that described in WO97/02357.

In the above description the use of the platform has been described inapplication which excite and sense fluorescence. It will be appreciatedthat the platform can be used in arrangements where affinity reactionsare detected by changes of luminescence. It will be also appreciatedthat the platform can be used in arrangements where affinity reactionsare detected by changes in refractive index.

Platforms in accordance with the present invention can be used in manyapplications of which the following is a non-exclusive list.

Gene expression

Comics

Pharmacogenomics

Toxicogenomics

Toxicoproteomics

Genetics

Pharmacogenetics

Toxicogenetics

Exon/intron expression profiling

Human Leukocyte Antigens (OLA) typing

Analysis of splicing variants

Proteomics (on-chip protein assays)

Patient monitoring (drug, metabolites, and markers)

Point-of-care, “personalised medicine”

Diagnostics

on-chip 2d gels for proteomics or 2d separation in general

SNP (single nucleotides polymorphism), mini-sequencing

High Throughput Screening

Combinatorial chemistry

Protein-protein interaction

Molecular interaction

Chip-based protein-antibody and peptide interaction

Green fluorescent protein (GFP)

in-situ hybridisation

confocal microscopy

fluorescence correlation spectroscopy (FCS)

conventional microscopy

MALDI-TOF MS

1-25. (canceled)
 26. A platform for use in sample analysis, saidplatform having one or more sensing areas or regions, each for receivinga capture element or elements which when the platform is irradiated withcoherent light can interact to provide an indication of an affinityreaction, wherein each capture element includes two or more types ofcapture molecule. 27-29. (canceled)
 30. The platform of claim 26,including an optically transparent substrate having a first refractiveindex (n₁), a thin, optically transparent layer, formed on one surfaceof the substrate, said layer having a second refractive index (n₂) whichis greater than the first refractive index (n₁), said platformincorporating therein one or multiple corrugated structures comprisingperiodic grooves which define the one or multiple sensing regions, saidgrooves being configured such that coherent light incident on saidplatform is diffracted resulting in an abnormal high reflection of theincident light thereby generating an enhanced evanescent field at asurface of the one or multiple sensing regions; wherein the enhancedevanescent field interacts with luminescent material on or in thevicinity of the surface of the one or more of the sensing regions so asto produce a detectable luminescent signal.
 31. The platform of claim30, wherein the light incident upon the platform has a polarization thatis substantially perpendicular to the grooves in at least one sensingregion so as to give rise to TM excitation in the at least one sensingregion.
 32. A process according to claim 31 wherein the light beam isincident onto the substrate side of the platform.
 33. A processaccording to claim 31 wherein the light beam is incident onto theoptically transparent layer side of the platform.
 34. The platform ofclaim 30, wherein the light beam incident upon the platform has apolarization that is substantially parallel to the grooves in at leastone sensing region so as to give rise to TE excitation in the at leastone sensing region.
 35. A process according to claim 34 wherein thelight beam is incident onto the substrate side of the platform.
 36. Aprocess according to claim 34 wherein the light beam is incident ontothe optically transparent layer side of the platform.